The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland.

The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Article Figures & Data

Figures

Overview of next-generation sequencing, neoantigen prediction, and functional T-cell analyses. Whole-exome sequencing was performed on the pretreatment and postprogression tumor and matched normal samples. Exome data were applied in a neoantigen prediction pipeline that evaluates antigen processing, MHC binding, and gene expression to generate neoantigens specific to the patient's HLA haplotype. Truncal neoantigens were identified by correcting for tumor purity and ploidy, and the TCR repertoire was evaluated at baseline, at the time of response, and upon emergence of resistance. Putative eliminated neoantigens at the time of resistance were used to generate peptides and stimulate autologous T cells, followed by TCR next-generation sequencing. PBL, peripheral blood lymphocyte.

Mutation cellularity analyses for eliminated mutations in pretreatment and postprogression tumor samples. Mutation cellularities at baseline (T1) and upon progression (T2) were estimated with the SCHISM pipeline; a cellularity of 0 was observed for 18, 10, 7, and 6 sequence alterations in resistant T2 tumors for CGLU116, CGLU117, CGLU127, and CGLU161, respectively (A). These somatic mutations were lost either by LOH or by subclonal elimination at the time of emergence of therapeutic resistance to immune checkpoint blockade. Somatic mutations in SLC26A7, PGAP1, HELB, and ANKRD12 that are associated with functionally validated neoantigens were detected in the pretreatment tumors but not in the resistant tumor or matched normal DNA; MAF denotes the mutant allele frequency (B).

Emergence of resistance to immune checkpoint blockade is associated with elimination of mutation-associated neoantigens by LOH and a more diverse T-cell repertoire independent of PD-L1 expression. A, CT images of patient CGLU117 at baseline, at the time of therapeutic response, and at time of acquired resistance. Pretreatment CT image of the abdomen demonstrates a right adrenal mass (T1, circled). Radiologic tumor regression is noted after 2 months of treatment, followed by disease relapse at 4 months from treatment initiation with a markedly increased right adrenal metastasis (T2, circled). Third follow-up CT demonstrates further disease progression in the adrenal lesion. Tumor burden kinetics for target lesions by RECIST criteria are shown in B. Peripheral T-cell expansion of a subset of intratumoral clones was noted to peak at the time of response and decrease to baseline levels at the time of resistance (C). Productive TCR frequency denotes the frequency of a specific rearrangement that can produce a functional protein receptor among all productive rearrangements. D and E, B allele frequency graphs for chromosome 17. A value of 0.5 indicates a heterozygous genotype, whereas allelic imbalance is observed as a deviation from 0.5. The region that undergoes LOH in the resistant tumor (E, orange box) contains three mutation-associated neoantigens that are thus eliminated. No differences in CD8+ T-cell density (F and G) or PD-L1 expression (H and I) were observed between baseline and resistant tumors.